Enzymes have been widely utilized as sensitive labels in a number of biochemical systems, including immunoassays such as ELISA systems, and nucleic acid assays such as PCR and sequencing systems. The enzymes are often detected indirectly, based on their activity, typically based on their transformation of substrate into product, or of a co-factor between, e.g., oxidized and reduced states.
In some implementations, the enzyme to be detected is attached to a highly specific complexing or binding agent such as an antibody. When the antibody binds to a target molecule to be detected, the antibody complex can be detected by observing the presence of the enzyme label attached to it; the enzyme is readily detected based on its activity. In other systems, an oligonucleotide to be expressed is labeled by linking it to a nucleotide that encodes an enzyme that can function as a label. When the oligonucleotide is expressed, the protein product that includes the enzyme label, which facilitates detection, again based on the activity of the enzyme.
Detecting the activity of the enzyme provides very efficient signal amplification. Rather than detecting the often small amount of enzyme (or target compound) present, one looks for the activity of the enzyme, i.e., its effect on known substrates that can be added in relatively large amounts. A single enzyme molecule can catalyze transformation of a large number of substrate molecules (e.g., an enzyme may catalyze 107 reactions per minute: THE IMMUNOASSAY HANDBOOK, 3rd ed. by David Wild, Elsevier Press, pg. 194 (2005)), so the species actually detected can be a product formed by the enzyme or the disappearance of a substrate or co-factor consumed by the enzyme, instead of the enzyme itself. Thus when observing the enzyme's activity, one detects large numbers of substrate or product molecules rather than the enzyme itself, which provides a highly amplified signal.
A number of such enzyme labels are known: the ones most commonly used in immunoassays (e.g., ELISA) include horseradish peroxidase and alkaline phosphatase. Others that have been used include acetate kinase, firefly luciferase, xanthine oxidase, beta-D-galactosidase, glucose oxidase, and glucose 6-phosphate dehydrogenase. Id. at 194-195.
However, there remains a need for new methods to label biochemical species to facilitate detection of extremely small quantities, and novel enzyme labeling systems are thus needed. There also remains a need for methods to detect trace amounts of hydrolytic enzymes in other settings where they are not used just as labels. The present invention provides such methods as well as compounds and compositions for use in these methods and conditions.